SOX5 promotes cell growth and migration through modulating DNMT1/p21 pathway in bladder cancer

Bladder cancer (BC) is one of the most prevalent and life-threatening cancers among the male population worldwide. Sex determining region Y-box protein 5 (SOX5) plays important roles in a variety of human cancers. However, little research has been conducted on the function and underlying mechanism of SOX5 in BC. In the present study, we first reveal the increased expression of SOX5 in BC tissues and in vitro cells lines. Second, we discover that inhibition of SOX5 inhibits cell growth and migration but promotes cell apoptosis. Meanwhile, ectopic SOX5 expression stimulates cell growth and migration in BC cells. Then, we show that suppressing SOX5 inhibits the expression of DNA methyltransferase 1 (DNMT1), and that overexpressing DNMT1 alleviates the cell progress of BC cells inhibited by SOX5. Furthermore, we demonstrate that DNMT1 inhibits p21 expression by affecting DNA methylation of the p21 promoter. Collectively, we demonstrate that SOX5 exerts its functions in BC cells by modulating the SOX5/DNMT1/p21 pathway. Finally, we demonstrate that SOX5 knockdown inhibits xenograft tumor growth in vivo. In conclusion, our study elucidates the oncogenic role of SOX5 and its underlying molecular mechanism in BC, and reveals a novel pathway which has the potential to serve as a diagnostic biomarker and therapeutic target for BC.


Introduction
Bladder cancer (BC) is one of the most prevalent and life-threatening cancers among the male population worldwide [1]. Currently, BC has the highest incidence of urinary system tumors and has a significantly higher mortality rate in China [2]. Despite significant advances in diagnosis and treatment, the recurrence rate of BC patients remains around 75%, resulting in a low level of fiveyear overall survival rate [3]. Furthermore, the biomarkers and molecular mechanisms underlying the development and progression of BC need to be investigated further.
SOX5, a transcription factor in the Sex determining region Y-box (SOX) family, is involved in a variety of cellular processes, including cell growth, cell apoptosis, cell cycle, cellular senescence, aging, and metabolism [4]. Furthermore, SOX5 has been linked to physiological and pathological processes in a variety of cancers, in-cluding breast cancer, gastric cancer, lung cancer, ovarian cancer, colorectal cancer, and hepatocellular carcinoma [5][6][7]. Previously, we discovered that silencing of SOX5 inhibited cell progress in BC [8]. However, the biological roles and the precise underlying regulatory mechanism of SOX5 in BC remain unknown.
In the process of DNA methylation, a methyl group from S-adenosylmethionine is transferred to the C-5 position of cytosines residing in CpG dinucleotides [9]. In mammals, three types of DNA methyltransferases (DNMTs), i.e., DNMT1, DNMT3a, and DNMT3b, catalyze DNA methylation [10]. The most abundant DNMT is DNMT1 which maintains DNA methylation patterns in newly synthesized DNA, whereas DNMT3a and DNMT3b methylate fully unmethylated CpG sites [11,12]. DNA methylation has an impact on a variety of cellular processes, including chromosome stability, chromatin structure, genomic imprinting, transcription, X chromosome inactivation, embryonic development, cell growth and death, and so on [12]. DNA methylation plays a significant role in the physiological and pathological progression of human diseases such as imprinting disorders, repeat-instability diseases, and cancers [13]. Many cancers have been shown to have oncogenic DNMT1-mediated DNA methylation [14]. DNMT1 has been linked to the physiological and pathological progression of BC [15,16]. Nevertheless, the upstream and downstream regulatory factors and mechanisms of DNMT1 in BC require further investigation.
As a downstream target of the well-known tumor suppressor p53, the cyclin-dependent inhibitor p21 inhibits cell growth and migration but promotes cell apoptosis and cell senescence [17]. Several studies focused on the inhibition of p21 expression by DNMT1mediated DNA methylation [18,19]. Another study focused on the role of p21 in the regulation of DNMT1 expression [20]. However, the relationship between DNMT1 and p21 in BC has not been studied.
Based on previous studies, we focused on investigating the function and the underlying mechanism of SOX5 in BC. We demonstrated that increased SOX5 expression in BC promotes DNMT1 expression, resulting in p21 inhibitory expression and, ultimately, the development and progression of BC.

Clinical specimens
All tumor tissue samples and adjacent normal tissues (located > 5 cm from tumors) were collected under the protocols approved and supervised by the Research Ethics Committee of the Xiangya Hospital, Central South University (Ethics Approval number: 202103548). All the tissues were immediately snap-frozen in liquid nitrogen. Informed consent agreements (written in the light of the Ethical Guidelines) were obtained from all patients. Patients' clinical information was listed in Table 1.
The sequences of SOX5, DNMT1 and p21 were synthesized by Generay (Shanghai, China) and inserted into the pCDNA3.1 vector. The plasmids were transfected into the cells using Lipofectamine-2000 (Invitrogen, Carlsbad, USA) according to the manufacturer's instructions. After 6 h of transfection, the medium containing the transfection reagent was replaced with fresh medium. Forty-eight hours after transfection, the cells were collected for further study.
Quantitative real-time polymerase chain reaction (qRT-PCR) Liquid nitrogen was used to grind the tissue samples. Trizol reagent (Junxin Biotech, Suzhou, China) was used to isolate the total RNA from tissues specimens or cells according to the manufacturer's instructions. PrimeScriptTM RT Master Mix (TaKaRa, Dalian, China) was applied to perform the reverse transcription according to the protocols. SYBR Green I qPCR Mix (Junxin Biotech) was used for quantitative real-time PCR with the ABI 7500 real-time PCR system (Applied Biosystems, Foster City, USA). The 2 -ΔΔCT method was introduced to calculate the relative expression levels of genes which were normalized to that of β-actin. Primers used for real-time PCR were designed using the US National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/) and synthesized by Sangon Biotech. The sequences of these primers are shown in Table 3.

Western blot analysis
The clinical specimens were ground with liquid nitrogen. Then, RIPA lysis buffer was introduced to extract the proteins from clinical specimens and cells. The protein concentration was quantified using a BCA detection kit (Junxin Biotech). Protein extract (30 μg) was separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred onto a nitrocellulose membrane (Millipore, Billerica, USA). Membranes were blocked with 5% skim milk, incubated with primary antibodies and horseradish peroxidase (HRP)-labeled secondary antibody successively. The protein bands were visualized using an ECL reagent (Junxin Biotech) on Gel Imagine System (Bio-Rad, Hercules, USA). Antibodies used in this study are shown as follows: anti-DNMT1 (Abcam, Cambridge, UK; ab87656), anti-SOX5 (Millipore; 07e131) and anti-p21 (Abcam; ab109520). The secondary antibody was purchased from Service Biotechnology Co., Ltd (Wuhan, China). The dilution ratio of antibodies was 1:1000.

Cell viability assay
Cell Counting Kit-8 (CCK8; Jinxin Biotech) was used to determine cell viability according to the manufacturer's instructions. For CCK8 analysis, the cells were planted into 96-well plates (4×10 3 cells/ well). After different treatments, the cells in each well were added with 10 μL of CCK8 regent. After incubation at 37°C for 2 h, absorbance was detected at OD450 nm using a micro-plate reader (Thermo Scientific, Waltham, USA).

Cell proliferation assay
The 5-ethynyl-2′-deoxyuridine (EdU) kit (Junxin Biotech) was used to determine cell proliferation according to the manufacturer's instructions. Before the EdU analysis, the cells were changed with fresh medium containing 200 μL of EdU regent, and grew at 37°C for 2 h. After being washed with 1 ×PBS buffer, the cells were fixed with 4% paraformaldehyde (PFA). Then, the cells were permeabilized with PBS containing 0.3% Triton X-100. Ultimately, the cells were stained with Apollo and Hoechst succes-sively. The stained cells were observed under a fluorescence microscope (ZKX53; Olympus, Tokyo, Japan). Red signals were EdU-stained cells, representing proliferating cells and meanwhile, and the blue signal was the nucleus, representing the overall cell number.

Cell migration assay
The Transwell chamber (Corning, Corning, New York, USA) was used to measure the migration ability of cells according to the manufacturer's instructions. Briefly, the cells were suspended in medium supplemented with 1% FBS before being seeded into the upper chamber (10,000 cells/well), and then the insert was added to the 24-well plate which was filled with 600 μL medium supplemented with 10% FBS. Twenty-four hours later, the cells in the chamber were removed with cotton swabs, and the migrated cells in the outer membrane of the chamber were stained with 0.1% purple crystal at room temperature for 30 min. After the air-drying, the migrated cells were observed under a microscope (ZKX53) and the number of migrated cells was counted.

Cell apoptosis assay
Apoptosis in NRCMs was determined by a TdT-mediated dUTP Nick-End Labeling (TUNEL) kit (Roche, Basel, Switzerland) based on the operation instructions. After being washed with 1 ×PBS buffer, the cells were fixed with 4% paraformaldehyde (PFA) and permeabilized with PBS containing 0.3% Triton X-100. Then, the cells were incubated with 50 μL TUNEL detection regents at 37°C for 1 h in the dark, and stained with Hoechst solution for 15 min at 37°C in the dark. The stained cells were observed under a fluorescence microscope (ZKX53). Green signals were TUNEL-stained cells, representing apoptotic cells, and meanwhile, the blue signal was the nucleus, representing the overall cell number.

Methylation-specific PCR (MSP) analysis
The Genomic DNA was extracted using the Genomic DNA Mini Preparation kit with Spin Column (Beyotime, Shanghai, China) according to the manufacturer's protocol. Genomic DNA was modified by EZ DNA methylation-GOLD kit (ZYMO Research, Irvine, USA) according to the manufacturer's instructions. The primers for the MSP are listed in Supplementary File 1.

Chromatin Immunoprecipitation (ChIP) assay
ChIP analysis was carried out according to the previously described method [21]. The antibodies used in ChIP analysis were listed below: anti-DNMT1 (Abcam; ab87656), and anti-SOX5 (Millipore; 07e131). The DNA of ChIP samples was isolated with the Genomic DNA Mini Preparation kit with Spin Column (Beyotime) according to the manufacturer's protocol, and the enrichment of DNMT1 promoter was measured using quantitative real-time PCR. The primers for DNMT1 promoter were: DNMT1-promoter F: 5′-CCCCCACACACTGGGTATAG-3′ and DNMT1-promoter R: 5′-AGGGTTTGTGAGAGCCCTTG-3′.

Generation of xenografts
Five week-old female athymic nude mice were obtained from the Model Animal Research Institute of Nanjing University (Nanjing, China). The right flanks of mice were injected subcutaneously with T24 cells (1×10 7 cells in 0.2 mL sterile PBS). The volume of xeno- Reverse CTTCTGACCCATACCCACCA 989 SOX5/DNMT1/p21 pathway in BC grafts was measured every week using calipers and calculated as follows: tumor volume=(length×width 2 )/2. The mice were euthanized one month after injection, and the xenografts were collected for further analyses. All animal experimental procedures were approved by the Committee on the Ethics of Animal Experiments of the Xiangya Hospital, Central South University.

Statistical analysis
Experiments were performed in triplicate and data were analyzed using GraphPad Prism 5.0 (GraphPad Software Inc., La Jolla, USA) and expressed as the mean±SD. Student's t-test was applied to assess the statistical differences. P<0.05 represents a statistically significant difference.

SOX5 is up-regulated in BC tissues and in vitro cell lines
To investigate the role of SOX5 in BC, human BC tissues as well as paired adjacent norm tissues were collected, and qPCR and western blot analysis were used to measure the SOX5 expression in these tissues. The results showed that SOX5 expression in BC was higher than that in normal tissues, both at the RNA and protein levels ( Figure 1A,B).
Then SOX5 expressions in human BC cell lines 5673, J82, T24, EJ-M3, and SVHUC-1 bladder epithelial cells were determined. Results showed that SOX5 expression was elevated in BC cell lines, as it was in BC tissues, when compared to bladder epithelial cells ( Figure 1C, D). These findings suggest that SOX5 may play a role in the progression of BC.

SOX5 positively regulates the cell process of BC cells
To investigate the role of SOX5 in cancer progression, we first created a SOX5 overexpression plasmid (pcDNA-SOX5) based on the pcDNA-3.1 vector, then synthesized siRNAs against SOX5 (SOX5-siRNAs), and transfected them into T24 and J82 cells. First, we used qPCR and western blot analysis to detect SOX5 mRNA and protein expression levels respectively and discovered that pcDNA-SOX5 significantly increased SOX5 expression, while SOX5-siRNAs significantly inhibited its expression in both cell lines (Figure 2A-C). In the subsequent experiments, only the results of siSOX5-1 were shown. The cell viability, proliferation, migration, and apoptosis of BC cells were investigated by the CCK8, EdU, Transwell, and TUNEL assays, respectively. SOX5 overexpression increased cell viability, proliferation, and migration in both T24 and J82 cells. Meanwhile, SOX5 knockdown had the opposite effect on the cellular processes of BC cells ( Figure 2D-F and Supplementary Figure S1A,B). siSOX5 induced apoptosis in BC cells, but no differences in apoptosis were found between the pcDNA-NC and pcDNA-SOX5 groups ( Figure 2G and Supplementary Figure S1C). All these results revealed that the knockdown of SOX5 can inhibit the cell viability, proliferation and migration, and it can also pro-

SOX5 promotes DNMT1 expression in BC cells
Because DNMT1 has been linked to BC [22], we wanted to know if SOX5 could influence DNMT1 expression in BC cells. We first transfected BC cells with pcDNA-SOX5, pcDNA-NC, SOX5-siRNAs, and NC-siRNAs. We detected DNMT1 expression after 48 h, and the results showed an increased DNMT1 expression in cells transfected with pcDNA-SOX5, compared to cells transfected with pcDH-NC ( Figure 3A,B,E), and an inversely decreased DNMT1 expression in  Figure 3C-E). ChIP analysis revealed that SOX5 could bind to the DNMT1 promoter, indicating that SOX5 regulates DNMT1 expression at the transcriptional level ( Figure 3F,G). These findings demonstrate that SOX5 increases DNMT1 expression in BC cells.

Ectopic expression of DNMT1 rescues the cellular processes caused by SOX5 knockdown in BC cells
We overexpressed DNMT1 (pcDNA-DNMT1) in wild-type cells or in SOX5-knockdown T24 and J82 cells to demonstrate if it mediates SOX5's role in BC cells. qPCR and western blot analysis revealed that pcDNA-DNMT1 significantly increased DNMT1 expression in wild-type cells ( Figure 4A-C). CCK8, EdU, Transwell, and TUNEL analysis revealed that DNMT1 overexpression reversed cellular processes in BC cells caused by SOX5 knockdown (Figure 4D-G and Supplementary Figure S1D-F), implying that SOX5's role in BC cells is mediated by DNMT1.

DNMT1 inhibits p21 expression in BC cells
It was reported that DNMT1-mediated DNA methylation regulated

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SOX5/DNMT1/p21 pathway in BC p21 expression in cancer cells [23]. To investigate the regulatory relationships between DNMT1 and p21 in BC cells, we treated the cells for 24 h with 5-AzadC at various concentrations (0, 0.5, 1.0, and 2.0 μM). qPCR and western blot analysis revealed that 5-AzadC significantly increased p21 expression in both cell lines ( Figure 5A-C). Then, in BC cells, we knocked down and overexpressed DNMT1. As expected, pcDNA-DNMT1 decreased p21 expression in both cells, whereas DNMT1-siRNAs significantly increased its ex- The cell proliferation, migration and apoptosis were measured by EdU assay, Transwell assay and TUNEL assay, respectively. The cells were divided into three groups: pcDNA-DNMT1+siSOX5, siSOX5 and siNC. β-Actin was used as the loading control. **P<0.01. Experiments were repeated three times. 993 SOX5/DNMT1/p21 pathway in BC pression ( Figure 5D-H). Methylation-specific PCR analysis revealed that pcDNA-DNMT1 increased the DNA methylation stage of the p21 promoter, whereas DNMT1-siRNAs inhibited the DNA methylation stage of the p21 promoter ( Figure 5I). These findings demonstrate that DNMT1 inhibits p21 expression in BC cells.

SOX5/DNMT1/p21 pathway regulates cell processes of BC cells
We used three groups of cells, i.e., pcDH-DNMT1+siSOX5, pcDH-NC+siSOX5, and pcDH-NC+siNC, to investigate the upstream and downstream regulatory relationship between DNMT1 and p21 in the function of SOX5 in BC. qPCR and western blot analysis revealed that pcDNA-DNMT1 reversed the up-regulation of p21 expression caused by SOX5-siRNAs in both cells ( Figure 6A,B), indicating that SOX5 silencing increased p21 expression by downregulating DNMT1 expression.
To investigate the role of the SOX5/DNMT1/p21 pathway in the cellular processes of BC cells, we first constructed a p21 over-

Knockdown of SOX5 suppresses the growth of xenograft tumors in vivo
Given the importance of SOX5 in BC cells, we investigated whether SOX5 affects the growth of xenograft tumors in vivo. T24 cells with stable knockdown of SOX5 were intraperitoneally injected into mice. The results showed that knockdown of SOX5 inhibited the growth of xenograft tumors in vivo ( Figure 7A-C). Furthermore, the expressions of SOX5 and DNMT1 were reduced, while p21 expression was increased in xenograft tumors treated with siSOX5 ( Figure 7D). These findings confirm that suppressing SOX5 inhibits the growth of xenograft tumors in vivo.

Discussion
BC is a common urological tumor and one of the most common male malignant tumors. Despite advances in diagnosis, treatment and experience, the 5-year survival rate for BC remains very low [24]. A better understanding of tumor characteristics aids in the development of new diagnostic and therapeutic approaches. The hallmarks of tumors are summarized by Douglas Hanahan and Robert A. Weinberg, which include self-sufficiency in growth signals, insensitivity to antigrowth signals, resistance to cell death, limitless replicative potential, sustained angiogenesis, tissue invasion and metastasis, avoiding immune destruction, tumor promotion inflammation, deregulating cellular energetics, genome instability, and mutation [25,26]. As a result, the essence of tumor prevention and treatment is to inhibit these ten tumor characteristics. Nal-

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SOX5/DNMT1/p21 pathway in BC trexone can activate the PI3K/AKT signaling pathway in BC [27]. LINC00355 was discovered to promote BC cell proliferation, invasion, and resistance to cisplatin [28,29]. All of these previous studies have revealed potential targets for promoting BC development.
In our study, we discovered that the SOX5/DNMT1/p21 pathway plays an important role in the cellular processes of BC, such as cell viability, proliferation and migration, indicating the potential application of this pathway in the diagnosis and treatment of BC. In vertebrates, the SOX family contains more than 20 genes divided into eight groups [5]. The SOX family, as an important group of transcription factors, plays important roles in cell growth and apoptosis, cell cycle, metastasis, epithelial to mesenchymal transition (EMT), metabolism, and tumorigenesis' physiological and pathological progression [5]. SOX5 has been linked to breast cancer, gastric cancer, colorectal cancer, lung cancer, and osteosarcoma [30][31][32][33][34]. SOX5 also regulates a variety of targets in cancers, including EZH2, twist 1, P2RY8, MITF, and others [6,7,35,36].
However, there has not been a lot of research done in BC on the function of SOX5 and its underlying molecular mechanism. SOX5 was found to be up-regulated in BC in our study. Further research revealed that SOX5 knockdown inhibited cell growth and migration in BC cells, whereas SOX5 overexpression promoted these cellular processes. Furthermore, silencing of SOX5 inhibited tumor growth in vivo. These findings provide preliminary support for SOX5's oncogenic role in BC.
DNA methylation is a common mode of gene expression regulation. As the primary DNA methylation transferase, DNMT1 plays a critical role in tumorigenesis. The oncogenic role of DNMT1 in a variety of cancers, including BC, has received considerable attention [37]. Nonetheless, no research has been conducted to demonstrate the regulatory relationships between SOX5 and DNMT1 in BC. Therefore, we investigated whether SOX5 plays a role in BC by regulating DNMT1. Our findings showed that SOX5 promoted DNMT1 expression, and that ectopic expression of DNMT1 reduced the role of inhibited SOX5 in the cellular functions of BC cells.
DNMT1 inhibits tumor suppressor genes in several cancers by hyper-methylating gene promoters [38]. Sanaei et al. [39] discovered that DNMT1 repression increased p21 expression in the colon cancer SW480 cell line. Statins, according to Dongoran et al. [40], had an anti-proliferative effect in OSCC cells by inhibiting DNMT1-regulated p21 expression. As a result, we are curious to know if DNMT1 regulates p21 in BC. Our findings first confirmed DNMT1's inhibitory regulation of p21 in BC cells. More excitingly, overexpression of DNMT1 reversed the elevation of p21 caused by silenced SOX5, implying that SOX5 silencing down-regulates DNMT1 expression, leading to the elevation of p21 expression in BC cells.
In conclusion, we elucidated the role and molecular mechanism of SOX5 in BC. Our findings showed that SOX5 expression was increased in BC tissues and in vitro cell lines, confirming SOX5's oncogenic role in BC. Our study revealed that increased SOX5 expression promoted DNMT1 expression and inhibited p21 expression via DNA methylation, indicating a potential role for the SOX5/ DNMT1/p21 pathway in the diagnosis and treatment of BC.

Supplementary Data
Supplementary data is available at Acta Biochimica et Biophysica Sinica online.

Funding
This work was supported by the grants from the Youth Science Foundation of Xiangya Hospital, Central South University (No. 2019Q09), the Natural Science Foundation of Changsha (No. kq2014274), the Natural Science Foundation of Hunan Province (Nos. 2020JJ5916, 2022JJ40826, and 2021JJ31082) and the National Natural Science Foundation of China (No. 82103298).